Vaccinia virus (VV), long considered the archetypal poxvirus, has long been a tool in biomedical research and vaccination purposes, and several recombinant poxvirus constructs are undergoing scientific review for various vaccine purposes. Vaccinia is considered representative of the other poxviruses.
Recombinant poxviruses have been generated by in vivo homologous recombination using a broad number of selection markers. See, for example, Mackett et al., Proc. Nat'l Acad. Sci. USA 79: 7415–19 (1982). Another approach for producing recombinant poxviruses is known as “direct molecular cloning.” See U.S. Pat. Nos. 6,103,244 and 5,445,953, both of which are incorporated by reference. Falkner et al., Ency. Life Sci. 1–4 (2001) and Moss, Proc. Nat'l Acad. Sci. USA 93: 11341–48 (1996) provide overviews of poxvirus technology.
Frequently-used procedures for generating recombinant poxviruses employ (i) thymidine kinase (tk)-negative selection by insertional inactivation of the endogenous vaccinia tk-gene, (ii) color screening using the E. coli β-galactosidase gene and (iii) dominant positive selection using the E. coli hypoxanthine-guanine phosphoribosyl-transferase (gpt) marker. These approaches allow for appropriately stringent selection for standard constructions with replicating parental poxviruses.
One major in vivo application for poxviruses is the use of vaccinia as a smallpox vaccine. This vaccine has led to the elimination of smallpox as a naturally-occurring disease. World events, such as the increasing threat of bioterrorism, have raised the specter of smallpox being employed as a biological weapon. See Dove, Nature Medicine 8: 197 (2002). Accordingly, large scale smallpox vaccination of populations is under active consideration.
Past vaccination programs have shown that the conventional smallpox vaccine is not without risk. See Lane et al., New Eng. J. Med. 281:1201–1208 (1969); Lane et al., J. Infect. Dis. 122:303–309 (1970). There are several adverse events, including diseases, associated with the conventional smallpox vaccine. These events are described below.
Postvaccinial Encephalitis
Postvaccinial encephalitis, although occurring infrequently, is one of the most serious adverse events associated with conventional smallpox vaccines. This adverse event is manifested by severe demyelinating encephalitis. The case-fatality rate is approximately 30 to 35%. Neurological sequelae are frequent with this adverse event, and include cognitive deficits and paralysis.
Post-vaccinal encephalitis typically is a complication of primary vaccination. The maximum risk of post-vaccinal encephalitis in adults following primary vaccination appears to be in the range of 3 to 9 affected patients per million vaccinated adults.
Progressive Vaccinia (vaccinia necrosum)
Progressive vaccinia is a serious adverse event usually seen only in immunosuppressed persons, such as those with hereditary or acquired immunodeficiency disorder, or undergoing treatment with immunosuppressive medications.
Progressive vaccinia is characterized by failure of the primary cutaneous lesion to heal. Symptoms include progressive enlargement and spreading, necrosis of the lesion, and appearance of other lesions, which in turn progress. The case-fatality rate is high (40 to 85%), with death generally occurring 2 to 5 months after vaccination.
The incidence of progressive vaccinia has been low (1 to 3 affected patients per million vaccinated subjected). This complication was more frequent in adults than children, and reflected the presence of tumors of the reticuloendothelial system (lymphoma or leukemia). HIV/AIDS, however, represents a risk factor for progressive vaccinia that was not seen during the era of wide-spread smallpox vaccination.
Eczema vaccinatum
Eczema vaccinatum is a serious adverse event that occurs in individuals with active or quiescent eczema, and is characterized by the appearance of cutaneous lesions in skin. Large areas of skin usually become involved, and there are severe systemic effects, such as fever and lymphadenitis. The case fatality rate is less than 10%.
Generalized Vaccinia.
Generalized vaccinia can occur in immunocompetent persons. Affected persons develop a generalized rash following vaccination. This illness is characterized by multiple skin lesions resembling the local reaction at the vaccination site, and can be accompanied by fever and chills. The highest incidence of this complication occurs in children who are less than 1 year of age.
Ocular Complications
Accidental infection of the eye of a contact results in infection of the eyelid, conjunctiva and, in some cases, the cornea. Corneal involvement (keratitis) is the most serious complication, occurring principally in persons undergoing primary vaccination. Keratitis is relatively infrequent, occurring in less than 10% of cases with ocular infection. The incidence of ocular vaccinia was approximately 10 affected patients per million vaccinated subjects.
The diagnosis of these adverse events is recognizable by the healthcare practitioner. Past treatment approaches have included the use of vaccinia immune globulin (VIG), but the overall effectiveness of these interventions with diseases resulting from conventional smallpox vaccines is uncertain.
Current Practices
Classical vaccinia strains are being increasingly replaced by highly attenuated and/or nonreplicating viruses for safety reasons. These replacement viruses are restricted to a relatively narrow host range. The nonreplicating vectors MVA (modified vaccinia Ankara) or NYVAC (New York vaccinia virus, derived from the Copenhagen strain), for instance, are propagated in primary chicken embryo fibroblasts. Although nonreplicating vaccinia vectors are safe, they often do not induce sufficient amounts of neutralizing antibodies when used as a single vaccine.
Other approaches for increasing safety include the use of poxviruses having disrupted or inactivated essential regions, typically through the insertion of foreign DNA into the essential regions. These viruses are permissive for growth only when complemented, such as through the use of engineered complementing cell lines. See, for example, U.S. Pat. No. 5,766,882 (which is incorporated by reference); Holzer et al., J. Virol. 71:4997–5002 (1997).
The vaccinia tk-gene, mentioned above for use in creating recombinant poxviruses, has exhibited a high tendency for spontaneous mutations. These mutations render the tk selection approach leaky (susceptible to the generation of tk-negative viruses without a foreign DNA insert). Thus, more stringent negative selectable markers are needed to permit better screening approaches and facilitate identification of essential poxviral genes. A clear identification of essential genes still depends on conditionally lethal mutations that can be mapped to a respective locus. Deletions enforced by a previously inserted dominant negative marker, should discriminate between essential and non-essential genes, even when no temperature sensitive mutants exist for that locus.
Vaccinia virus, both native and recombinant strains, can be inhibited by a variety of compounds. See De Clerc, Clin. Microbiol. Rev. 14:382–397 (2001). However, no drug is currently approved for treatment of generalized vaccinia infection, and only few drugs would be acceptable for treatment of humans according to their respective spectrum of side-effects. Drugs known to inhibit vaccinia efficiently, such as bromodeoxyuridine (BrdU), are not selective and inhibit also growth of the host cells. The thiosemicarbazones, which are potent inhibitors of vaccinia and smallpox virus, seem to be toxic to cells and are not approved medications for any indication. The most promising anti-vaccinia drug currently appears to be cidofovir, an antiviral approved for cytomegalovirus (CMV) infection. Cidofovir has been shown to reduce mortality in immunocompetent mice when challenged with cowpox virus; although it only delayed but did not prevent death of SCID mice. Cidofovir is administered by the intravenous route. Cidofovir treatment, however, requires hospitalization of patients because nephrotoxicity has to be prevented by infusions of normal saline before and after drug infusions. In addition, monitoring of kidney function is required, thereby making cidofovir treatment an expensive and sophisticated treatment option.
Other antiviral drugs, such as Azidothymidine (AZT), have been shown to inhibit replication of human immunodeficiency virus (HIV) do not normally inhibit vaccinia virus.
There are anti-viral alternatives to AZT. The control of HIV spread in a cell culture or an organism by other approaches has been described. Smith et al., Proc. Nat'l Acad. Sci. USA 93: 7955–7960 (1996) disclosed an effective vaccine approach for human HIV-1 employing a live-attenuated virus, wherein the virus is modified to express the herpes simplex virus (HSV) tk gene. The spread of HIV-1 in tissue culture is shown to be controllable by the addition of the drug gancyclovir (GCV). Chakrabarti et al., Proc. Nat'l Acad. Sci. USA 93: 9810–9815 (1996) described a candidate live attenuated vaccine for AIDS comprising a genetically modified HIV-1 virus comprising a HSV-tk gene as a controllable conditional lethal marker. Because the HSV-tk gene confers on the virus a preference of the prodrugs GCV and Acyclovir (ACV) over its normal substrate thymidine or guanine, the addition of the prodrugs permits control of the virus load in infected individuals whom have received the vaccine. However, the approach has risks due to the possible generation of escape mutants caused by the high mutation rate of HIV.
Recombinant vaccinia has been engineered for uses other than in vaccines. Recombinant vaccinia that confers drug sensitivity to tumor cells can be used as a cancer gene therapy approach. See Higginbotham et al. (12th Poxvirus Meeting, USA 1998). In addition, Metzger et al. J. Virol. 68: 8423–8427 (1994) disclosed a recombinant vaccinia comprising the coding sequence for cytomegalovirus (CMV) UL97 gene, which makes vaccinia susceptible to GCV. In this report, vaccinia is used as a tool to characterize a herpes virus tk gene encoded by ORF UL97. McChart et al. Gene Therapy 7:1217–1223 (2000) describes replicating viruses for cancer gene therapy, in particular a recombinant vaccinia expressing the cytosine deaminase gene, which converts the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). 5-FU has an anti-tumor effect and also an anti-viral effect.